Antibodies typically comprise two heavy chains linked together by disulfide bonds and two light chains linked to a respective heavy chain by a disulfide bond. Beginning at one end of each heavy chain there is a variable domain followed by several constant domains. Similarly, each light chain has a variable domain at one end, but only a single constant domain at its other end. There are two types of light chain, which are termed lambda (λ) and kappa (κ) chains. No functional difference has been found between antibodies having λ or κ light chains. The ratio of the two types of light chain varies from species to species, however. In mice, the κ:λ ratio is 20:1, whereas in humans it is 2:1.
The variable domains of the light and heavy chains are aligned, as are the constant domain of the light chain and the first constant domain of the heavy chain. The constant domains in the light and heavy chains are not involved directly in binding the antibody to antigen.
It is the variable domains that form the antigen binding site of antibodies. The general structure of each light and heavy chain domain comprises a framework of four regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs). The four framework regions employ a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site.
While cell surface antigens of tumor cells are the traditional targets for antibody-guided cancer therapy, one of the major limitations for the therapy of solid tumors is the low accessibility of tumor antigens to antibodies circulating in the blood stream. The dense packing of tumor cells and the elevated interstitial pressure in the tumor core present formidable physical barriers.
A solution to the problem of poor penetration of antibodies into solid tumors would be to attack the endothelial cells lining the blood vessels of the tumor rather than the tumor cells themselves. While it may be difficult to target the mature tumor vasculature specifically, i.e., without destroying healthy tissue, promising strategies aim at the inhibition of neovascularization.
Neovascularization, also termed angiogenesis, is induced by cytokines that are secreted from tumor cells and depends on vascular cell migration and invasion, processes regulated by cell adhesion molecules (CAM) and protease. These molecules are currently considered potential targets for angiogenic inhibitors. In this regard, the vascular integrin αVβ3 has recently been identified as a marker of angiogenic blood vessels. See Brooks, P. C., et al. (1994), REQUIREMENT OF INTEGRIN αVβ3 FOR ANGIOGENESIS, Science 264, 569–571. Moreover, it was shown that the mouse monoclonal antibody (Mab) LM609 directed to integrin αVβ3 was able to suppress angiogenesis, indicating than integrin αVβ3 has a critical role in angiogenesis.
It has been further demonstrated that LM609 selectively promotes apoptosis of vascular cells that have been stimulated to undergo angiogenesis. See Brooks, P. C., et al. (1994), INTEGRIN αVβ3 ANTAGONISTS PROMOTE TUMOR REGRESSION BY INDUCING APOPTOSIS OF ANGIOGENIC BLOOD VESSELS, Cell 79, 1157–1164. These findings suggest that integrin αVβ3 may be a target and LM609 a tool for cancer diagnosis and therapy.
Indeed, LM609 not only prevented the growth of histologically distinct human tumors implanted on the chorioallantoic membranes of chicken embryos, but also induced their regression. See, Cell 79, 1157–1164. Using a more clinically relevant model of tumor growth, it was found that LM609 blocked human breast cancer growth in a SCID mouse/human chimeric model. Importantly, not only did LM609 block tumor growth, but it also inhibited metastasis of the breast carcinomas examined. See Brooks, et al. (1995) ANTI-INTEGRIN αVβ3 BLOCKS HUMAN BREAST CANCER GROWTH AND ANGIOGENESIS IN HUMAN SKIN, J. Clin. Invest. 96, 1815–1822.
The Brooks et al. results are consistent with previous studies that have suggested that angiogenesis contributes to the metastatic spread of breast tumor cells. See Weidner, N., et al. (1991) TUMOR ANGIOGENESIS AND METASTASIS: CORRELATION IN INVASIVE BREAST CARCINOMA, N. Engl. J. Med. 324, 1–8; and Weidner, N., et al. (1992) TUMOR ANGIOGENESIS: A NEW SIGNIFICANT AND INDEPENDENT PROGNOSTIC INDICATOR IN EARLY-STAGE BREAST CARCINOMA, J.Natl. Cancer Inst. 84, 1875–1887.
Within the last few years evidence has been presented that two cytokine-dependent pathways of angiogenesis exist and that these are defined by their dependency on distinct vascular integrins. See Friedlander, M., et al. (1995) DEFINITION OF TWO ANGIOGENIC PATHWAYS BY DISTINCT AV INTEGRINS, Science 270, 1500–1502. The results of the Friedlander et al. studies show that anti-αVβ3 antibody LM609 blocked angiogenesis in response to bFGF and TNFα, yet have little effect on angiogenesis induced by VEGF, TGFα, or phorbol ester PMA. In contrast, the anti-αVβ5 antibody P1F6 blocks angiogenesis induced by VEGF, TGFα, and phorbol ester PMA, while having minimal effects on that induced by bFGF or TNFα.
It is conceivable, thus, that tumors showing less susceptibility to anti-αVβ3 antibodies might secrete cytokines that promote angiogenesis in an αVβ5-dependent manner. Taken together, both anti-αVβ3 and anti-αVβ5 antibodies are promising tools for diagnosis and therapy of cancer.
Mouse monoclonal antibodies such as LM609, however, are highly immunogenic in humans, thus limiting their potential use for cancer therapy, especially when repeated administration is necessary. To reduce the immunogenicity of mouse monoclonal antibodies, chimeric monoclonal antibodies were generated, with the variable Ig domains of a mouse monoclonal antibody being fused to human constant Ig domains. See Morrison, S. L., et al. (1984) CHIMERIC HUMAN ANTIBODY MOLECULES; MOUSE ANTIGEN-BINDING DOMAINS WITH HUMAN CONSTANT REGION DOMAINS, Proc. Natl. Acad. Sci. USA 81, 6841–6855; and, Boulianne, G. L., et al. (1984) PRODUCTION OF A FUNCTIONAL CHIMAERIC MOUSE/HUMAN ANTIBODY, Nature 312, 643–646. This process is commonly referred to as “humanization” of an antibody.
In general, the chimeric monoclonal antibodies retain the binding specificity of the mouse monoclonal antibody and exhibit improved interactions with human effector cells. This results in an improved antibody-dependent cellular cytotoxicity which is presumed to be one of the ways of eliminating tumor cells using monoclonal antibodies. See Morrison, S. L. (1992) IN VITRO ANTIBODIES: STRATEGIES FOR PRODUCTION AND APPLICATION, Ann. Rev. Immunol. 10, 239–265. Though some chimeric monoclonal antibodies have proved less immunogenic in humans, the mouse variable Ig domains can still lead to a significant human anti-mouse response. See Bruggemann, M., et al. (1989) THE IMMUNOGENICITY OF CHIMERIC ANTIBODIES, J. Exp. Med. 170, 2153–2157. Therefore, for therapeutic purposes it may be necessary to fully humanize a murine monoclonal antibody by altering both the variable and the constant Ig domains.
Full humanization is feasible by introducing the six CDRs from the mouse heavy and light chain variable Ig domains into the appropriate framework regions of human variable Ig domains. This CDR grafting technique (Riechmann, L., et al. (1988) RESHAPING HUMAN ANTIBODIES FOR THERAPY, Nature 332, 323) takes advantage of the conserved structure of the variable Ig domains, with the four framework regions (FR1–FR4) serving as a scaffold to support the CDR loops which are the primary contacts with antigen. U.S. Pat. No. 5,502,167 to Waldmann, et al. discloses a “humanised antibody” having the CDR loops LCDR1 through LCDR3 and HCDR1 through HCDR3 from YTH 655(5)6, a rat IgG2b monoclonal antibody, grafted onto a human T cell antibody.
A drawback, however, of the CDR grafting technique is the fact that amino acids of the framework regions can contribute to antigen binding, as well as amino acids of the CDR loops can influence the association of the two variable Ig domains. To maintain the affinity of the humanized monoclonal antibody, the CDR grafting technique relies on the proper choice of the human framework regions and site-directed mutagenesis of single amino acids aided by computer modeling of the antigen binding site (e.g., Co, M. S., et al. (1994) A HUMANIZED ANTIBODY SPECIFIC FOR THE PLATELET INTEGRIN gpllb/lla, J. Immunol. 152, 2968–2976). A number of successful humanizations of mouse monoclonal antibodies by rational design have been reported. Among them are several monoclonal antibodies that are directed to human integrins and have potential clinical application. See, J. Immunol. 152, 2968–2976; Hsiao, K. C., et al. (1994) HUMANIZATION OF 60.3, AN ANTI-CD18 ANTIBODY; IMPORTANCE OF THE L2 LOOP, Protein Eng. 7, 815–822; and, Poul, M. A., et al. (1995) INHIBITION OF T CELL ACTIVATION WITH A HUMANIZED ANTI-BETA 1 INTEGRIN CHAIN mAb, Mol. Immunol. 32, 101–116.
Human immunoglobulin transgenic mice provide a promising alternative to the humanization of mouse monoclonal antibodies. See, e.g., Fishwild, D. M., et al. (1996) HIGH-AVIDITY HUMAN IgGK MONOCLONAL ANTIBODIES FROM A NOVEL STRAIN OF MINILOCUS TRANSGENIC MICE, Nature Biotechnology 14, 845–851. In response to immunization, these mice express human monoclonal antibodies, which can be accessed by conventional hybridoma technology.
Rational design strategies in protein engineering have been challenged by in vitro selection strategies that are mainly based on phage display libraries. See Clackson, T., and Wells, J. A. (1994) IN VITRO SELECTION FROM PROTEIN AND PEPTIDE LIBRARIES, TIBTECH 12, 173–184. In particular, in vitro selection and evolution of antibodies derived from phage display libraries has become a powerful tool. See Burton, D. R., and Barbas III, C. F. (1994) HUMAN ANTIBODIES FROM COMBINATORIAL LIBRARIES, Adv. Immunol. 57, 191–280; and, Winter, G., et al. (1994) MAKING ANTIBODIES BY PHAGE DISPLAY TECHNOLOGY, Annu. Rev. Immunol. 12, 433–455.
The development of technologies for making repertoires of human antibody genes, and the display of the encoded antibody fragments on the surface of filamentous bacteriophage, has provided a means for making human antibodies directly. The antibodies produced by phage technology are produced as antigen binding fragments—usually Fv or Fab fragments—in bacteria and thus lack effector functions. Effector functions can be introduced by one of two strategies: The fragments can be engineered either into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.
Typically, the Fd fragment (VH-CH1) and light chain (VL-CL) of antibodies are separately cloned by PCR and recombined randomly in combinatorial phage display libraries, which can then be selected for binding to a particular antigen. The Fab fragments are expressed on the phage surface, i.e., physically linked to the genes that encode them. Thus, selection of Fab by antigen binding co-selects for the Fab encoding sequences, which can be amplified subsequently. By several rounds of antigen binding and reamplification, a procedure termed panning, Fab specific for the antigen are enriched and finally isolated.
In 1994, an approach for the humanization of antibodies, called “guided selection”, was described. Guided selection utilizes the power of the phage display technique for the humanization of mouse monoclonal antibody. See Jespers, L. S., et al. (1994) GUIDING THE SELECTION OF HUMAN ANTIBODIES FROM PHAGE DISPLAY REPERTOIRES TO A SINGLE EPITOPE OF AN ANTIGEN, Bio/Technology 12, 899–903. For this, the Fd fragment of the mouse monoclonal antibody can be displayed in combination with a human light chain library, and the resulting hybrid Fab library may then be selected with antigen. The mouse Fd fragment thereby provides a template to guide the selection.
Subsequently, the selected human light chains are combined with a human Fd fragment library. Selection of the resulting library yields entirely human Fab.
For the full humanization of murine monoclonal antibodies, the present invention uses a unique combination of CDR grafting and guided selection. The anti-integrin antibody generated is useful for cancer diagnosis and therapy.